Kynureninases are a group of pyridoxal-5'-phosphate dependent enzymes which catalyze the hydrolytic .beta.,.gamma.-cleavage of aryl-substituted .alpha.-amino-.gamma.-keto acids, particularly L-kynurenine or 3-hydroxy-L-kynurenine to give L-alanine and anthranilic acid or 3-hydroxyanthranilic acid, respectively (see: K. Soda and K. Tanizawa (1979) Advances Enzym. 49:1-40). Kynureninase is involved in the microbial catabolism of L-tryptophan via the aromatic pathway. In plants and animals, a kynureninase is required in tryptophan catabolism and for NAD biosynthesis via quinolinic acid. Quinolinic acid is a relatively toxic metabolite, and in particular, it is a potent neurotoxin which has been implicated in the etiology of neurological disorders, including epilepsy and Huntington's chorea (R. Schwarcz et al. (1988) Proc. Natl. Acad. Sci. USA 85:4079; M. F. Beal et al. (1986) Nature 321:168-171; S. Mazzari et al. (1986) Brain Research 380:309-316; H. Baran and R. Schwarcz (1990) J. Neurochem. 55.:738-744). It has been found that the activity of this pathway for tryptophan matabolism in mammals is elevated by interferon and by transient ischemia (Ozaki et al. (1988) Proc. Natl. Acad. Sci. USA 85: 1242-1246; Saito et al. (1993) J. Neurochem. 60: 180). The resultant increase in brain and/or serum quinolinate may result in neuropathology. Inhibitors of kynureninase are thus important targets for treatment of neurological disorders, including strokes. High levels of nicotinylalanine, a weak inhibitor of kynureninase in vitro, have been shown to inhibit the accumulation of quinolinic acid in vivo, following administration of bacterial endotoxins to rats (Moroni et al. (1991) J. Neurochem. 57: 1630).
L-kynurenine (which can also be designated .alpha.,2-diamino-.gamma.-oxobenzenebutanoic acid) is the preferred substrate of bacterial kynureninase, which is exemplified by that of Pseudomonas fluorescens (O. Hayaishi and R. Y. Stanier (1952) J. Biol. Chem. 195:735-740). The kynureninase of tryptophan metabolism in plants and animals has a somewhat different substrate specificity with 3-hydroxy-L-kynurenine (which can be designated .alpha.,2-diamino-3-hydroxy-.gamma.-oxobenzenebutanoic acid) being the preferred substrate (Soda and Tanizawa (1979) supra).
The mechanism of kynureninases has been the subject of considerable interest due to the unique nature of this pyridoxal-5'-phosphate dependent reaction. Mechanisms based on redox reactions (J. B. Longenecker and E. E. Snell (1955) J. Biol. Chem. 213:229-235) or transamination (C. E. Dalgleish et al. (1951) Nature 168:20-22) have been proposed. More recently mechanisms involving either a nucleophilic mechanism with an "acyl-enzyme" intermediate (C. Walsh (1979) "Enzymatic Reaction Mechanisms" W. H. Freeman and Co., San Francisco, p. 821; M. Akhtar et al. (1984) "The Chemistry of Enzyme Action," New Comprehensive Biochemistry, Vol. 6 (M. I. Page, ed.) Elsevier, New York, p.821) or a general base-catalyzed mechanism (K. Tanizawa and K. Soda (1979) J. Biochem. (Tokyo) 86:1199-1209) have been proposed. Kynureninase is now recognized as one of only a few pyridoxal-5'-phosphate (PLP) dependent enzymes which catalyze reactions with electrophilic substitution at the .beta.-carbon. In contrast, many PLP-dependent enzymes catalyze reactions involving nucleophilic substitution at the .beta.-carbons of amino acids. The unique feature of the proposed mechanism of kynureninase is the resonance stabilized alanine .beta.-carbanion intermediate generated by the retro-Claisen cleavage of the gem-diol of kynurenine [Phillips and Dua (1991) J. Am. Chem. Soc. 113: 7385; Dua et al. (1993) J. Am. Chem. Soc. 115: 1264].
In addition to the physiological reaction, kynureninase has been shown to catalyze an aldol-type condensation of benzaldehyde with incipient L-alanine formed from L-kynurenine to give .alpha.-amino-.gamma.-hydroxy-.gamma.-phenylbutanoic acid (G. S. Bild and J. C. Morris (1984) Arch. Biochem. Biophys. 235:41-47). The stereochemistry of the product at the .gamma.-position was not determined, although the authors suggested that only a single isomer was formed.
J. L. Stevens (1985) J. Biol. Chem 260:7945-7950 reports that rat liver kynureninase displays cysteine conjugate .beta.-lyase activity. This enzyme activity is associated with cleavage of S-cysteine conjugates of certain xenobiotics to give pyruvate, ammonia and a thiol, for example, cleavage of S-2-(benzothiazolyl)-L-cysteine to give 2-mercaptobenzothiazole, pyruvate and ammonia. More recently, I. S. Blagbrough et al. (1990) Toxicol. Lett 53(1-2):257-259 (Chem. Abstract 114(9):77537k) report that cysteine conjugate .beta.-lyase (C-S-lyase) is a member of a family of transaminases and aminotransferases and that C-S lyase is a glutamine transaminase K. The reference discusses structure-activity relations displayed by C-S-lyases. C-S-lyases are distinguishable from kynureninase but exhibit overlapping activities.
Several reports concerning the relative reactivities of kynurenine analogs with bacterial kynureninase or rat liver kynureninase are summarized in Soda and Tanizawa (1979) supra. Tanizawa and Soda (1979) supra reported that a number of ring substituted L-kynurenines, namely: 3-hydroxy-, 5-hydroxy-, 5-methyl-, 4-fluoro-, and 5-fluoro-L-kynurenine were substrates of kynureninase of P. fluorescens. These authors also reported that dihydrokynurenine (called .gamma.-(o-aminophenyl)-L-homoserine therein) was a substrate for that kynureninase, yielding o-aminobenzaldehyde and L-alanine. The K.sub.m of dihydrokynurenine was reported to be 67 .mu.M compared to a K.sub.m of 35 .mu.M for L-kynurenine and 200 .mu.M for 3-hydroxy-L-kynurenine. N'-formyl-L-kynurenine and .beta.-benzoyl-L-alanine were likewise reported to be substrates (with K.sub.m =2.2 mM and 0.16 mM, respectively) for the bacterial kynureninase. Tanizawa and Soda measured relative reactivity as relative amount of L-alanine formed.
O. Hayaishi (1955) in "A Symposium on Amino Acid Metabolism" (W. D. McElroy and H. B. Glass, eds.) Johns Hopkins Press, Baltimore, pp. 914-929, reported that 3-hydroxy- and 5-hydroxy-L-kynurenine, .beta.-benzoyl-L-alanine and .beta.-(o-hydroxybenzoyl)-L-alanine were substrates for the bacterial enzyme, but that N'-formyl-L-kynurenine was not a substrate. O. Hayaishi measured relative reactivities by determining the amounts of substrate hydrolyzed.
Tanizawa and Soda (1979) supra reported that S-benzoyl-L-cysteine, L-asparagine and D-kynurenine were not substrates of kynureninase, while O. Hayaishi (1955) Supra reported that .beta.-(p-aminobenzoyl)-L-alanine, .beta.-(o-nitrobenzoyl)-L-alanine, .beta.-(m-hydroxybenzoyl)-L-alanine, 3-methoxy-L-kynurenine, .beta.-benzoylpropanoic acid, and .beta.-(o-aminobenzoyl)propanoic acid do not react with bacterial kynureninase. Kynureninase is reported to act only on L-amino acids (M. Moriguchi et al. (1973) Biochemistry 12:2969-2974).
O. Wiss and H. Fuchs (1950) Experientia 6:472 (see: Soda and Tanizawa (1979) supra) reported that 3-hydroxy-L-kynurenine, L-kynurenine, .beta.-benzoyl-L-alanine, .gamma.-phenyl-L-homoserine, .gamma.-methyl-L-homoserine, 2-aminolevulinic acid and .alpha.-amino-.gamma.-hydroxypentanoic acid reacted with rat liver kynureninase to produce alanine, while .beta.-(o-nitrobenzoyl)-L-alanine did not.
G. M. Kishore (1984) J. Biol. Chem. 259:10669-10674 has reported that certain .beta.-substituted amino acids are mechanism-based inactivators of bacterial kynureninase. Several .beta.-substituted amino acids including: .beta.-chloro-L-alanine, O-acetyl-L-serine, L-serine O-sulfate, S-(2-nitrophenyl)-L-cysteine (called S-(o-nitrophenyl)-L-cysteine, therein) and .beta.-cyano-L-alanine inactivated kynureninase. These .beta.-substituted amino acids react with kynureninase to give pyruvate and ammonia. Amino acids with good leaving groups at the .beta.-carbon, such as .beta.-chloroalanine, were shown to act as irreversible inactivators of kynureninase from P. marginalis. However, these compounds have high K.sub.i values and inactivate a large number of PLP-dependent enzymes. S-(2-nitrophenyl)-L-cysteine, which is not a .beta.-substituted amino acid, was described as the "most efficient suicide substrate at low concentrations" with a K.sub.i of 0.1 mM.
Bacterial kynureninase is also strongly inhibited by o-aminobenzaldehyde (K.sub.i =6.5 .mu.M, non-competitive inhibition). Several other aromatics having "a carboxyl group on the benzene ring and an amino group at the ortho-position" including o-aminoacetophenone, anthranilic acid o-nitrobenzaldehyde and benzaldehyde were described as inhibitors (Tanizawa and Soda (1979) supra). It was suggested that inhibition relates to binding of the formyl group to the portion of the enzyme that serves as a binding site for the .gamma.-carboxyl of kynurenine. Anthranilate and 3-hydroxyanthranilate, the products of the kynureninase reaction, were also reported to inhibit the enzyme (Takeuchi et al. (1980) J. Biochem. (Tokyo) 88:987-994).
Blagbrough, I. S. et al. (1988) Drug Metab. Drug Interact 6(3-4):303-316 in Chem. Abstracts 112(19):174617c report on inhibition of rat renal C-S lyase by certain cysteine conjugates. Certain S-(nitro-substituted phenyl)-L-cysteines and N-acetyl-S-(nitro-substituted phenyl)-L-cysteines were reported to inhibit C-S lyase as measured by a kidney slice methodology. The nitrophenyl cysteine conjugates: S-(2-nitrophenyl)-L-cysteine, S-(4-nitrophenyl)-L-cysteine, S-(2,6-dinitrophenyl)-L-cysteine, N-acetyl-S-(3,4-dinitrophenyl)-L-cysteine, N-acetyl-S-(2,6-dinitrophenyl)-L-cysteine and N-acetyl-S-(2-chloro-4-nitrophenyl)-L-cysteine are reported to inhibit C-S layse.
Vamvakas et al. (1988) Chem. Biol. Interact 65:59-71 in Chem. Abstracts 109(7):50020w refers to the cysteine conjugate .beta.-lyase-mediated metabolism of certain cysteine conjugates including S-benzyl-L-cysteine, which was reported to be cleaved to give pyruvate. The reference notes that aminooxyacetic acid is an inhibitor of the .beta.-lyase.
Tolosa et al. (1968) Mol. Biol. 2(5):769-777 [in Russian] in Chem. Abstracts 70(1):482d reported that cysteine lyase was significantly inhibited by H.sub.2 NOH and its O-substituted derivatives and that aminooxyacetic acid was the most inhibitory derivative tested.
J. P. Whitten et al. (1989) Tetrahedron Letts. 30:3649-3652 reported the synthesis of 2,2-difluoro-.alpha.-benzoyl alanine (.alpha.-amino-.beta.,.beta.-difluoro-.gamma.-oxobenzene butanoic acid) which is said to be a "potential new inhibitor of kynureninase." Fluoroketone-containing peptides are described as capable of forming stable hydrates or hemiketals which are "thought to inhibit" proteolytic enzymes as analogs of a tetrahedral transition state. The difluoro compound is described as a competitive inhibitor of kynureninase, but no details of this inhibition are given in the reference.
The present work is based on a reexamination of the mechanism of kynureninase catalysis, in particular, through an investigation of the stereospecificity of the retro-aldol reaction catalyzed by the enzyme. During the course of this work, the reactivity of dihydrokynurenine with kynureninase was found to be significantly different than had previously been reported. The result of these mechanism and reactivity studies was the identification of a class of potent kynureninase inhibitors. The present invention provides kynureninase inhibitors which are designed to be "transition-state analogue" inhibitors.